During the study on the oleandomycin production, we purified a new oleandomycin derivative having a macrolactone of which biosynthesis does not follow the genetic architecture of the oleandomycin PKS. The molecular formula for the compound was suggested as C35H59NO11 on the basis of the analysis of NMR and HRMS data (m/z 670.4185, Delta-1.9mmu, calcd for C35H60NO11). 13C NMR assignments and analysis of COSY, HMBC and HMQC data suggested that the compound differs from oleandomycin by formation of the olefinic functionality resulting from the dehydration of a hydroxy group in oleandomycin. The new oleandomycin derivative has antibacterial activities similar to those of oleandomycin agaisnt Enterococcus faecalis, Bacillus subtilis and Staphylococcus aureus.